Herpes simplex virus type 1 (HSV) infections of the cornea cause significant ocular morbidity and blindness in the U.S. HSV infections are a particular clinical problem because of the capacity of the virus to become latent and reactivate to re-initiate recrudescent disease. Traditionally, neurons of sensory ganglia have been characterized as the primary site of latency of HSV. Viral DNA is retained in neurons with limited transcription of viral genetic information. We and others have shown that, in addition to neurons, viral DNA can be maintained in the cornea during latency. As in neurons, some of this DNA has the ability to spontaneously reactivate in cell culture and produce infectious virus, suggesting that the DNA maintained in the cornea may be a source of virus for recrudescent disease. One aim of this study is to determine the state the viral DNA maintained in corneal cells of rabbits with a history of herpetic eye disease using pulsed field electrophoresis (PFE) to isolate the DNA. Amplification of selected viral DNA sequences using the polymerase chain reaction (PCR) and restriction enzyme analysis will help characterize its physical state. Free circular or linear viral DNA will be distinguished from viral DNA integrated into cell DNA. The DNA, especially that coding for viral transactivators, will be analyzed and modified to determine the effect of hypermethylation of promoter sequences on the expression. Origins of viral DNA replication will be studied using PCR to confirm the potential of the DNA to replicate. Studies using in situ PCR will help to determine whether corneal cells contain low numbers of copies among many cells or many copies of the viral genome in few cells. Marker rescue studies will determine whether the DNA retained is biologically active. Transcriptional activity of genes known to regulate virus replication as well as the gene characteristically transcribed in neuronal latency will be measured. Reverse PCR and immunocytochemical methods will be used to confirm that demethylation of key HSV genes actually results in transcription and translation. Corneas of rabbits will be infected with defective recombinant virus expressing reporter genes in order to determine whether HSV genes can be expressed in corneal cells which maintain non- replicating HSV DNA. Completion of the proposed studies will result in characterization of physical and transcriptional state of the DNA maintained in corneal cells during latency as well as assessment of its potential for reactivation.